Crystallographic data on the 27 kDa fragment of the methylcobalamin (Mecbl) dependent enzyme methionine synthase (MS) from E. Coli [C. L. Drennan et al. Science, 266, 1669 (1994)] and on the 5-deoxyadenosylcobalamin (Adocbl) dependent enzyme methylmalonyl-coenzyme A mutase (MMCoA) from Propionibacterium shermanii [F. Mancia et al. Structure, 1996, 4, 339.] show that in both enzymes the cofactor undergoes dramatic structural changes upon enzyme binding. The most significant change involves the dimethylbenzimidazole (DMB) group of the cofactor, which in both cases is detached and replaced by a His group of the enzymes. Both X-ray crystallographic structures have their limitations; the MS structure at 3 resolution is not being able to show distance variations in the cofactor structure in the order of 0.3 or less with respect to the free state and the MMCoA structure was collected on a dominantly Co(II) form of the cofactor lacking the adenosyl moiety. We have collected XAS data on both cofactors in their full enzyme bound states to examine the structural changes upon enzyme binding. Our results show that the structure of the cofactor stays the same in the Mecbl dependent enzyme MS both in the wild type Co(III)-methyl and in the Co(II) forms with respect to the free states but changes in the Adocl dependent MMCoA. The Co-N axial bond to the DMB of Adocbl in MMCoA is very long (2.45 ) similar to the crystallographic data which shows a 2.5 Co-N axial distance for the Co(II) state. Our EXAFS data for the Co-C bond shows two minima, one at around 1.99 and one at 2.25 . Based on recent RR data [R. Banerjee at al., unpublished results], change in the Co-N axial bond length does not affect the Co-C bond strength in MMCoA and so change of the Co-C bond length is not expected. Although the shorter distance is more likely based on the RR data, the longer Co-C distance cannot be ruled out by our EXAFS data. We have also examined mutant forms of MS to examine the role of the His759, Asp757, and Ser810 residues in the hydrogen bonding network. X-ray edge result (in agreement with EPR data) on the Co(II) form of the MS mutant H759G shows that replacing His759 with a Gly residue causes the formation of a four-coordinate species (identified by the 1s-4pz transition with no Gly ligation to the Co. This mutant shows five magnitude decrease in the catalytic activity with respect to the wild-type Co(II) (Matthews et al., Biochemistry, submitted). This is supported by our earlier result that a four-coordinate Co(II) corrinoid species is very unstable (Wirt et al., J. Am. Chem. Soc., 1993, 115, 5299 and Scheuring et al, J. Phys. Chem., 1996, 100, 3344). The EXAFS results also support a four-coordinate species with an average Co-Neq distance of 1.89+/-0.01 similar to other cobalamin species. The Co(II) form of mutant D757N, in which Asp757 is replaced by a Glu, indicates similar geometry to that of the wild-type Co(II). EPR data shows that this mutant is 70% bas-off and its activity is impaired by 50-fold. These results show that His759 in MS is essential to the catalytic activity and any change in the hydrogen bonding network decreases the activity substantially. Variations of the hydrogen bonding network below the corrin may account for the distinctive properties of cobalamin dependent enzymes and so similar studies are planned on different mutants of MMCoA, in which the hydrogen bonding network is lacking the Ser residue.